This invention relates to neurology and pharmacology, and to drugs for reducing brain damage in crisis situations such as stroke, cardiac arrest, drowning, or severe blood loss.
Neurons, especially in the central nervous system (CNS), can be severely damaged or killed by a condition called "excitotoxicity", which involves over-stimulation of neurons to a point where they begin dying. This condition arises during medical crises such as strokes, asphyxiation, carbon monoxide poisoning, cardiac arrest, internal hemorrhaging, severe blood loss, and various types of head injuries and other physical traumas. Certain types of poisons can also lead to excitotoxic brain damage. Seizures and convulsions due to epilepsy, head trauma, and various other causes also involve dangerous over-stimulation of neurons. Although relatively mild seizures are not presumed to cause neuronal death or permanent damage, severe seizures which cannot be halted by anti-seizure medications can cause permanent brain damage and neuronal death, due to excitotoxicity.
In addition, several types of progressive neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis) also are believed to involve excessive neurotransmitter activity as a component of the disease process.
These problems are severely aggravated by the inability of nerve cells to regenerate or repair themselves after injury. A child who suffers only a few minutes of perinatal asphyxia during birth may spend an entire lifetime horribly crippled by the injuries such damage can inflict. Many people who have suffered from strokes live the rest of their lives partially paralyzed, or unable to speak or remember major events in their lives due to permanent neuronal damage. Brain damage is an extremely serious medical problem which devastates individuals and their families, and inflicts enormous expense on insurance companies, government agencies, and others.
These costs are so high, largely because there currently are no effective drugs available for preventing or reducing brain damage during and after a crisis such as a stroke or cardiac arrest. Although stroke is the third leading cause of death in the United States, as of early 1997, not a single drug treatment has been approved by the U.S. Food and Drug Administration for preventing or reducing brain damage caused by stroke or any of the other causes of excitotoxic brain damage.
General Background on Neurons
The following is a very brief overview, intended mainly to direct the reader's attention to several cellular components and processes that are involved in this invention. For additional background information, a good single-volume textbook used at most medical schools is Principles of Neural Science, by Kandel & Schwartz (Elsevier Publishing, New York, 1996). Additional information is contained in G. Adelman's Encyclopedia of Neuroscience (Birkhauser Publishing, Boston), a multi-volume treatise, and in tens of thousands of scientific and medical articles, many of which are published in specialized journals such as Brain and Brain Research.
A neuron in the central nervous system (CNS) consists of a cell body, which contains the nucleus, and a strand-like projection called an axon, through which nerve impulses travel. The axon branches out into hundreds or even thousands of smaller fibrils, called synaptic processes. Each fibril terminates at a synaptic terminus, containing a small bulb-shaped area (a "bouton") which is bathed in extra-cellular synaptic fluid. The fluid fills the gap between the synaptic terminus (which is part of the "transmitting" neuron) and a receptor on a nearby "receiving" neuron.
Neurotransmitter molecules are stored at the end of each synaptic process, in synaptic boutons. One of the most important excitatory neurotransmitters in the mammalian CNS is glutamate, the ionized form of glutamic acid, an amino acid. Aspartate (the ionized form of aspartic acid, another amino acid) is also used by the brain as an excitatory neurotransmitter, but to a much lesser extent. Since glutamate and aspartate are amino acids, they are often called "excitatory amino acids", and glutamate receptors (discussed below) are sometimes called "excitatory amino acids receptors" (or EAA receptors).
When a nerve impulse reaches the end of a glutamate-containing axon, the glutamate molecules stored in the end of the axon are released into the extracellular synaptic fluid. These glutamate molecules temporarily bind to and react with glutamate receptors, which are proteins on the surface of the adjacent signal-receiving neuron (these proteins straddle the cell membrane, so that a portion of the membrane is exposed on the cell surface).
This brief binding reaction, between a glutamate molecule and a glutamate receptor, triggers a complex set of events in the signal-receiving neuron. The major steps in this reaction include: (1) opening of an ion channel associated with the receptor; (2) inflow of positively-charged calcium and sodium ions into the neuron, through the opened channel; (3) depolarization of the neuron, caused by the entry of charged calcium and sodium ions into the neuron.
This "depolarization" of a neuron by an incoming nerve signal (in the form of a glutamate molecule contacting a glutamate receptor on the neuron surface) is regarded as a triggering event, which takes the glutamate-triggered neuron into a brief state of hyperactivity. In order to be ready to receive an incoming nerve signal, neurons constantly try to maintain themselves in a polarized condition, i.e., in a condition where a relatively large electrical voltage potential (typically about -70 millivolts, mV) exists across the cell membrane, due to steep gradients of certain ions such as calcium. To maintain this polarization level, neurons pump calcium ions (Ca.sup.++) and sodium ions (Na.sup.+) outside the cell. This pumping action is so strong that the concentration of calcium ions outside a neuron, in the extracellular fluid that bathes a neuron, is roughly 10,000 times higher than the concentration inside the neuron.
In other words, the "resting" state of a neuron is a condition where the neuron is, in effect, sitting on top of an energy plateau. The neuron is ready to fire, in a manner comparable to a spring-loaded gun, where the spring is fully depressed and the mechanism is cocked, so that the gun is ready to fire a bullet as soon as the trigger is pressed. As soon as an incoming nerve signal arrives, the cell quickly depolarizes and triggers the sequence of events that lead to release of its own neurotransmitters. In effect, when this depolarization/firing occurs, the neuron comes down off of its high-energy, ready-to-fire plateau. Within a few milliseconds after that, the neuron begins working (and expending energy) to pump out calcium and sodium ions again, to regain its polarized resting state, so that it will be ready to receive the next nerve impulse.
There are also other ion gradients that exist across neuronal membranes, due to both active pumping and passive diffusion. Potassium ions (K.sup.+) are pumped inside neurons, but that pumping system is relatively weak. Chloride ions (Cl.sup.-) are driven out of a neuron, but this is due to the electronegativity of the fluid inside a resting cell, and is not believed to be due to an ion-specific pump.
The depolarizing flow of calcium and sodium ions into a neuron also causes other cellular responses at other locations (often called "downstream" locations) on the neuron. If the triggering event is sufficiently strong to overcome the effects of various inhibitory neurotransmitters (such as dopamine, serotonin, or GABA), then the depolarizing event will cause the neuron to release some of its own glutamate molecules at one or more downstream synaptic terminals, thereby passing the nerve signal on to other neurons, which then undergo similar depolarizing activation events as they pass on the nerve signal(s) to still other neurons.
Glutamate neurotransmitter molecules do not permanently bond to glutamate receptors at a synaptic junction. Instead, the glutamate molecules quickly disengage from the glutamate receptors and return to the synaptic fluid. Under normal and healthy conditions, the glutamate molecules which have been released by the receptors are rapidly pumped back into the neurons (or into glial cells, which effectively act as support cells inside the brain), by a glutamate transport system, which requires energy to carry out its pumping actions. This prevents glutamate from accumulating in the synaptic gaps between neurons, where it might cause excess stimulation of signal-receiving neurons.
However, in crisis conditions such as stroke or cardiac arrest, the transport system which normally removes the glutamate from the synaptic fluid runs out of energy, and can no longer function properly. When this happens, excess glutamate begins to accumulate in the synaptic gaps between neurons. This can quickly lead to a toxic condition, where the presence of lingering glutamate in the synaptic junctions causes severe and possibly continuous overstimulation (excitation) of the glutamate receptors.
This type of uncontrolled activation by glutamate is a key factor in "excitotoxicity" in the brain. It can lead to rapid and dangerous cellular deregulation, and it can severely aggravate and expand the amount of permanent brain damage that is suffered by a victim. By way of illustration, in many stroke victims, the central area of damaged brain tissue (which died because it lost its blood supply) is often surrounded and accompanied by a substantial "penumbra" of dead or dying neurons which were not directly affected by the loss of blood supply. Even though they were not directly affected by a cutoff of their blood supply, the dead or dying neurons in this penumbra region were, in effect, dragged into a toxic cascade, in which dangerously overstimulated neurons began releasing uncontrollable amounts of glutamate. Under excitotoxic conditions, the same glutamate molecules which play an essential role as neurotransmitters, in a healthy brain, can become deadly toxins. When this condition occurs, the glutamate molecules begin to unleash neurotoxic processes that can quickly lead to the deaths of penumbral neurons that are outside the region of brain tissue that was directly injured by a loss of blood flow.
Ischemia, Hypoxia, and Neuron Damage
The requirement that neurons must very rapidly pump out calcium and sodium ions to regain a "ready to fire" status within a few milliseconds after an activation spike leads to a number of biochemical factors that help explain how and why the brain and spinal cord can be so rapidly and badly damaged, during and after an ischemic or hypoxic crisis.
The conditions which most commonly cause brain damage are referred to by physicians and researchers as ischemia (which refers to lack of adequate blood flow) and hypoxia (which refers to inadequate oxygen supply). Ischemia occurs in the brain during a stroke, cardiac arrest, severe blood loss due to injury or internal hemorrhage, and other similar conditions that disrupt normal blood flow. It also occurs after a head trauma that causes "edema" (fluid accumulation which leads to swelling of soft tissue) inside the brain, since the pressure caused by edema presses against and flattens the arteries and veins inside the brain, thereby reducing their ability to carry blood through the brain.
Hypoxia also can be caused in various ways. It is a direct result of ischemia; whenever blood supply is cut off, oxygen supply is also cut off as a direct result. However, hypoxia can occurs in various other conditions, even if blood flow remains unaltered; examples include carbon monoxide poisoning, drowning, suffocation, and other forms of asphyxia.
Hypoglycemia (an inadequate supply of glucose in the blood, which can occur due to conditions such as malnutrition, or an overdose of insulin in a diabetic) is less common, but it is also a substantial medical problem. All discussion herein relating to the use of calcium channel blockers to prevent or reduce excitotoxic brain damage is also applicable to preventing or reducing brain damage caused by hypoglycemia.
Because of certain physiological factors (e.g., neurons have no reserve supplies of glucose or oxygen), the brain and spinal cord are much more vulnerable to ischemic or hypoxic damage than any other organ, and permanent brain damage (including neuronal death) begins to occur within a few minutes. Because of their crucial roles in the body, damage to the brain or spinal cord can be quickly lethal, or can inflict permanent crippling damage and utter devastation to a victim's life.
Neurologists and other researchers have spent billions of dollars trying to develop drugs that can prevent or reduce ischemic or hypoxic damage to the brain and spinal cord. Although a number of approaches appear to hold promise for the future, the sad and tragic fact is that, as of early 1998, not a single type of drug which can effectively reduce or prevent excitotoxic brain damage due to stroke, cardiac arrest, asphyxiation, blood loss, or similar crises, is available to people who need such help; the only arguable exception is clot-dissolving drugs, such as streptokinase and tissue plasminogen activator, which can help dissolve blood clots, but which do not otherwise block or reduce the processes involved in glutamate excitotoxicity.
A great deal of neurological research on efforts to reduce ischemic/hypoxic brain damage has focused on synaptic receptors, especially glutamate receptors, because of the role glutamate accumulation plays in excitotoxicity. One of the primary theories behind this research is that if drugs can be used to prevent excessively accumulating glutamate from contacting glutamate receptors in the synaptic junctions between neurons, then the signal-receiving neurons will not be so vulnerable to toxic over-stimulation.
However, since glutamate is an essential neurotransmitter, global blockade of glutamate receptors can impose severe disruptions on proper and necessary neurological functioning, and can cause dangerous and potentially brain-damaging or even lethal side effects. Researchers studying drugs that can selectively block certain subclasses of glutamate receptors (these subclasses include NMDA receptors, kainic acid receptors, and AMPA receptors) have been claiming for years that these drugs can reduce brain damage in ischemia or hypoxia. However, most of those drugs have toxic side effects, and despite the claims of the researchers, no such drugs are available for public use--not even for critically ill patients who are dying of massive strokes or cardiac arrest.
Accordingly, attention by some researchers has recently turned toward various other avenues, including methods of reducing ion flow through the calcium channels that allow calcium ions to enter neurons during a depolarizing (activating) event. This field of research is discussed below.
Calcium Channels
Detailed information on neuronal calcium channels (and on various drugs that can selectively block calcium entry through different classes of calcium channels) is contained in articles such as Bertolino and Llinas 1992, Olivera et al 1994, Dunlap et al 1995, and Wheeler et al 1996.
Briefly, neurons possess at least four (and possibly more) types of calcium channels, located in their plasma membranes. The three classes which were known by the mid-1980's are called N, L, and T channels (Nowycky et al 1985). More recently, a fourth class called P channels has been widely recognized. Other classes, tentatively called the O, Q, and R channels, have also been suggested, but they are not yet widely agreed upon and identified consistently by all neuroscientists.
Based on various published reports and on original research by the Inventors herein, it is believed that P-type and Q-type calcium channels belong to a "P/Q" family. They are distinct from each other in certain ways, but they share a relatively high degree of homology and cross-reactivity (also called cross-affinity). Because of their relatively high levels of homology and cross-reactivity, P-type and Q-type channels respond similarly to various types of drugs, apparently including the radial poly-guanidino drugs that are the subject matter of this invention. Accordingly, references herein to "P/Q channels" are deemed to include references to either or both types of channels. Similarly, phrases such as "poly-guanidino drugs which can block P/Q channels" are intended to apply to radial poly-guanidino drugs as described herein which can block either P-type channels, or Q-type channels. To the best of the Applicants' knowledge and belief, all of the poly-guanidino compounds tested to date in tissue culture or in vivo tests (described below) block both P-type and Q-type calcium channels, as well as N-type calcium channels.
The four main classes of calcium channels (L, N, T, and P) can be distinguished from each other, in cell culture experiments, by the fact that certain drugs bind to the different classes of calcium channels with differing affinities. Certain dihydropyridine drugs (such as flunarizine, nicardipine, and nifedipine) bind to L-type channels, but not to T or N channels. Other fast-acting poisons called omega conotoxins (used in nature by marine snails of the genus Conus, to catch and paralyze fish) bind very tightly to N-channels, less tightly to L-channels, and even less tightly to T-channels (Kasai et al 1987). Certain types of spider toxins called agatoxins bind tightly to P channels (and possibly to Q-type channels as well).
All four of these classes of Ca.sup.++ channels exist on the surfaces of neurons, but not in the same locations. In neurons, L-type and T-type channels are located on the main body of a neuron, and on a neuron's dendrites (which are finger-like fibrils that carry arriving nerve impulses from a receptor-bearing synapse toward the main body of the neuron). Accordingly, both L and T channels can be regarded as post-synaptic channels; they are involved in how a neuron responds to a nerve impulse, after the impulse has arrived at an impulse-receiving synapse.
By contrast, N-type and P-type calcium channels are positioned "downstream" from the cell nucleus; they are positioned between the cell nucleus and a different set of synapses that will pass on nerve signals to other neurons. For this reason, N-type and P-type calcium channels are often called pre-synaptic calcium channels.
It is generally agreed among most researchers that calcium entry through N or P/Q channels is a necessary step in a series of neuronal actions that enable the release of glutamate (and, to a lesser extent, aspartate) from synaptic boutons as a neuron transmits a nerve signal to other neurons. Accordingly, conotoxins (from marine snails) or agatoxins (from spiders) which can selectively block the entry of calcium into neurons through N-type or P-type calcium channels can prevent the activated neurons from releasing glutamate. This effectively blocks the drug-treated neurons from transmitting the nerve signal to other neurons.
Drugs Disclosed in the Prior Art
The discoveries above, relating to pre-synaptic N and P/Q channels, have led to various efforts to develop drugs (modelled after the conotoxins or agatoxins) that can suppress glutamate release by excited neurons. Efforts to create peptide segments modelled after snail toxins, to suppress calcium entry via N-channels, are described in Patent Cooperation Treaty (PCT), number WO-91/07980 (invented by Miljanich et al, assigned to Neurex Corporation of Menlo Park, Calif.). Efforts to suppress calcium entry via P-channels, involving both peptide and non-peptide molecules modelled after spider toxins, are described in U.S. Pat. Nos. 4,925,664 (Jackson et al 1990), 4,950,739 (Cherksey et al 1990), 5,122,596 (Phillips et al 1992), and in numerous items of prior art cited therein.
Most of the spider toxins which can block P-channels contain large numbers of amine groups, in relatively small molecules. That realization led to the creation of various types of polyamines which assertedly can block calcium entry through P-channels. Such polyamines are described in, for example, U.S. Pat. Nos. 5,037,846 (Saccomano et al 1991); 5,227,397 (Saccomano et al 1993); and 5,242,947 (Cherksey et al 1993), and in various articles cited therein.
Various other efforts also have been made by other research teams to develop other drugs containing multiple amine groups, for neuroprotective purposes. In particular, Goldin et al (at Cambridge NeuroSciences) and Weber and Keana (at the University of Oregon and Oregon Health Sciences University) have each developed various amine compounds derived from guanidine, having the general structure: ##STR1## where the various R groups are selected from any number of organic moieties listed in various U.S. patents. The Goldin et al/Cambridge NeuroScience patents involving guanidine derivatives include U.S. Pat. Nos. 5,403,861 (April 1995); 5,438,130 (August 1995); 5,614,630 (March 1997); 5,622,968 (April 1997); 5,637,623 (June 1997); 5,652,269 (July 1997); 5,670,519 (September 1997); 5,672,608 (September 1997); 5,677,348 (October 1997); 5,681,861 (October 1997); and 5,686,495 (November 1997). Similar patents which involve guanidine derivatives and which list Weber and Keana as co-inventors include 4,709,094 (November 1987); 4,906,779 (March 1990); 5,093,525 (March 1992); 5,190,976 (March 1993); 5,262,568 (November 1993); 5,308,869 (May 1994); 5,312,840 (May 1994); 5,478,863 (December 1995); 5,502,255 (March 1996); 5,552,443 (September 1996); 5,559,154 (September 1996); 5,574,070 (November 1996); 5,604,228 (February 1997); and 5,637,622 (June 1997). As should be apparent from the dates, not all of these patents are prior art against the current invention, which was originally disclosed in an application filed in February 1996. However, as mentioned below, none of these patents are believed to involve arginine residues, and they do not involve radial branches that are evenly distributed around a central atom such as a tertiary amine or a benzene ring.
Weber, Keana, et al also have developed various bicyclic compounds derived from quinoline or quinoxaline-diones, as shown below: ##STR2## Such compounds are discussed in, for example, U.S. Pat. Nos. 5,475,007 (December 1995); 5,514,680 (May 1996); 5,597,922 (January 1997); 5,620,978 and 5,620,979 (April 1997); 5,622,952 (April 1997); 5,622,965 (April 1997); 5,631,373 (May 1997); and 5,652,368 (July 1997).
Another nitrogen-containing class of compounds that were identified as calcium channel blockers is described in U.S. Pat. No. 5,312,928 (Goldin et al, Cambridge NeuroScience, May 1994). These compounds are relatively complex molecules, having a bicyclic structure coupled via an amidine linkage to a linear nitrogen-containing structure.
To the best of the Applicant's knowledge and belief, none of the compounds listed in the above-cited US patents by Goldin et al or by Weber, Keana et al contain arginine residues. The only compounds known to the Applicant company which contain arginine residues, and which have been asserted to offer neuroprotective benefits by suppressing activity at calcium channels, are peptide (proteinous) molecules, in which multiple amino acids are coupled to each other in a linear chain via conventional peptide bonds (as used by cells to make proteins from amino acids). However, peptide drugs tend to cause substantial problems and suffer from other limitations when administered to humans, for various reasons including: (1) peptide drugs are readily broken down by the digestive system, if administered orally; and (2) foreign peptides can cause major problems by provoking immune rejection responses, if injected into a patient. Accordingly, most peptide drugs are strongly disfavored, if other non-peptide drugs can be identified and developed which have comparable useful activity without having a peptide structure.
Additional types of nitrogen-containing compounds are disclosed in U.S. Pat. No. 5,599,984 (Bianchi et al, Picower Institute, February 1997). Some of the compounds disclosed herein have radial structures, using a center nitrogen atom (such as compounds 31 and 34-36, in FIGS. 7G and 7H of the '984 patent) or a center benzene ring (such as compounds 28, 32, and 33, in FIGS. 7F and 7G). However, none of the compounds disclosed in the '984 patent used arginine residues. In addition, those compounds were not disclosed as being neuroprotective; instead, there were identified as being anti-inflammatory agents, and agents useful for suppressing arginine uptake, to help fight arginine-dependent tumors and infections.
Accordingly, despite all of the above-cited work (as well as decades of other research by thousands of other skilled neurological researchers), there are not yet available, to victims of stroke, cardiac arrest, asphyxiation, head trauma, or other medical crises that lead to ischemia or hypoxia in the brain, any compounds which can safely and effectively prevent or reduce excitotoxic brain damage. To the best of the Applicant's knowledge and belief, the compounds disclosed above all suffer from one or more limitations, such as cytotoxic side effects, low ability to permeate blood-brain barriers, difficulty in synthesis or purification, etc.
In addition, based on research carried out by the Applicant herein (Cypros Pharmaceutical Corporation), it appears that suppression of calcium entry at both N-type and P-type (and possibly Q-type) calcium channels may be more beneficial than selective blockade of only one class of pre-synaptic channels, in preventing or reducing excitotoxic damage to neurons.
Furthermore, the compounds disclosed herein have been shown to perform quite well in protecting brain tissue against ischemic or hypoxic damage, both in tissue culture tests, and in in vivo tests on live adult mammalian animals. In the in vivo tests on intact animals, these compounds penetrate blood-brain barriers and effectively protect the brain tissue against genuine ischemia, rather than merely against simulated ischemia, as used in cell culture tests.
Accordingly, one object of the subject invention is to disclose new compounds which can suppress calcium ion entry into neuron via pre-synaptic N-type and P/Q-type calcium channels.
Another object of the invention is to disclose new compounds which can suppress pre-synaptic calcium entry into neurons more effectively, by blocking both N-type and P/Q-type calcium channels rather than blocking only one class of pre-synaptic calcium channel.
Another object of this invention is to disclose drugs and methods which can help prevent or reduce excitotoxic damage to neurons, both in the CNS and in the peripheral nervous system.
Another object of this invention is to disclose improved methods of synthesizing drugs that can offer neuroprotective benefits during and after ischemic and hypoxic crises, by suppressing calcium ion entry into neurons through pre-synaptic calcium channels.
Another object of this invention is to disclose a new class of drugs useful for suppressing certain types of unwanted excessive neuronal activation, including neuropathic pain.
These and other objects of the invention will become more apparent through the following summary, drawings, and description of the preferred embodiments.